Antibodies are an important class of pharmaceutical products that have been successfully used in the treatment of various human diseases and conditions, including infectious diseases, cancer, allergic diseases, and graft-versus-host disease, as well as in the prevention of transplant rejection.
For many applications, it is desirable to alter the amino acid sequence of antibodies produced in particular animals. For example, one problem associated with the therapeutic application of non-human immunoglobulins is the potential immunogenicity of the same in human patients. In order to reduce the immunogenicity of such preparations, various strategies for the production of partially human (humanized) and fully human antibodies have been developed. One approach involves altering the sequence of the endogenous immunoglobulin locus in the antibody producing non-human animal in order to generate transgenic antibodies. The ability to produce transgenic antibodies having a human idiotype in non-human animals is particularly desirable as antigen binding determinants lie within the idiotype region, and non-human idiotypes are thought to contribute to the immunogenicity of antibody therapeutics. Human idiotype is an especially important consideration in respect of monoclonal antibody therapeutics, which consist of a single idiotype delivered at relatively high concentration as opposed to the variety of idiotypes delivered at lower concentrations by a polyclonal antibody mixture.
Attempts have been made to alter genomic sequences in cultured cells by taking advantage of the natural phenomenon of homologous recombination. See, for example, Capecchi (1989) Science 244:1288-1292; U.S. Pat. Nos. 6,528,313 and 6,528,314. If a polynucleotide has sufficient homology to the genomic region containing the sequence to be altered, it is possible for part or all of the sequence of the polynucleotide to replace the genomic sequence by homologous recombination. However, the frequency of homologous recombination under these circumstances is extremely low. Moreover, the frequency of insertion of the exogenous polynucleotide at genomic locations that lack sequence homology typically exceeds the frequency of homologous recombination by several orders of magnitude.
The introduction of a double-strand break into genomic DNA, in the region of the genome bearing homology to an exogenous polynucleotide, has been shown to stimulate homologous recombination at this site by several thousand-fold in cultured cells. Rouet et al. (1994) Mol. Cell. Biol. 14:8096-8106; Choulika et al. (1995) Mol. Cell. Biol. 15:1968-1973; Donoho et al. (1998) Mol. Cell. Biol. 18:4070-4078. See also Johnson et al. (2001) Biochem. Soc. Trans. 29:196-201; and Yanez et al. (1998) Gene Therapy 5:149-159. In these methods, DNA cleavage in the desired genomic region was accomplished by inserting a recognition site for a meganuclease (i.e., an endonuclease with a recognition sequence that is so large it does not occur, or occurs only rarely, in the genome of interest) into the desired genomic region.
Meganuclease cleavage-stimulated homologous recombination using naturally occurring meganucleases relies on either the fortuitous presence of, or the directed insertion of, a suitable meganuclease recognition site in the vicinity of the genomic region to be altered. Since meganuclease recognition sites are rare or nonexistent in a typical mammalian genome, and insertion of a suitable meganuclease recognition site is plagued with the same difficulties associated with other genomic alterations, methods employing conventional meganucleases have not found widespread use.
The engineering of meganucleases with novel specificities has expanded the applicability of meganuclease cleavage-stimulated homologous recombination. For example, see US 20050064474; and Moehle et al., PNAS, 104:3055-3060, 2007. However, many potential limitations to the application of cleavage-stimulated homologous recombination remain and may preclude its use in certain cell types, such as single cell mammalian embryos.
Mammalian embryos are highly sensitive to manipulation, and any damage incurred at this stage of development can have profound consequences. Accordingly, the use of cleavage-stimulated homologous recombination in mammalian embryos faces several obstacles. The introduction of nucleic acids into the pronuclei of embryos can be toxic, with embryo survival inversely correlated to the amount of nucleic acid introduced (For example, see “Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes.” ML Van Keuren, G B GGavrilina, W E Filipiak, M G Zeidler, T L Saunders. Transgenic Res. 2009 October; 18(5):769-85. Epub 2009 Apr. 26). In addition, cleavage of chromosomal DNA by meganucleases has the potential to cause chromosomal instability in any cell type.
Combining detrimental manipulations in mammalian embryos in particular could result in a high frequency of death, rendering cleavage-stimulated homologous recombination impracticable in embryos. The developmental program by which a mammalian embryo grows into a viable fetus is dependent on complex genetic and epigenetic processes involving hundreds, if not thousands, of genes and non-coding RNAs. The success of embryonic development is dependent on not only the proper quantitative regulation of each involved transcriptional unit, but their proper timing and coordinated expression. For a mammalian embryo which is embarking on the pathway to development into a viable fetus, it is not possible to extrapolate data on the effect or toxicity of nucleic acid introduction and chromosome cleavage in a cultured cell model to predict the effect on embryonic development.
Thus, the stress associated with chromosome cleavage and nucleic acid introduction suggests that cleavage-stimulated homologous recombination may be impracticable in embryos. In addition, success in gene targeting by homologous recombination may be subject to the epigenetic status of the locus containing the target site, which may vary between cell types and stages of development.